Epigenetic regulations participate in numerous biological processes and their errors have been implicated in many diseases including cancer. Among the key modulators in epigenetic are protein methyltransferases (PMTs). Strong evidence showed that PMTs function through methylating histone and nonhistone targets. Nonetheless, fully understanding the biological roles of PMTs is restricted by the inability to profile the PMT targets and to characterize the downstream functions. PMT-specific inhibitors can readily hijack PMT- involved epigenetics and thus serve as valuable chemical genetic tools to investigate these processes. However, the lack of high throughput screening (HTS) methods represents a significant barrier in identifying such small-molecule entities. The situation, if not addressed, greatly limits the application of chemical genetic tools to define, perturb and manipulate the epigenetic functions. The objective of this proposal is to develop generalizable HTS assays of PMTs for identifying target-specific PMT inhibitors. The critical analysis of PMTs structures formulated the central hypothesis that the distinct substrate/cofactor-binding pockets of PMTs can be exploited for developing target-specific inhibitors. The goal of this proposal will be achieved by pursing two specific aims. A 384-well mix-and-measure scintillation proximity assay (SPA) has been developed and is expected to be transformed into a 1536-well AlphaScreen format (Aim 1). These HTS assays will be then validated with a pilot compound library. With the aid of counter-screening and secondary assays (Aim 1), target-specific PMT inhibitors are expected to be unambiguously identified and characterized in vitro and in vivo (Aim 2). These inhibitors can be used to generate PMT-specific hypomethylation proteome for target profiling and to disrupt specific methylations for function characterization. In conjunction with two NIH Roadmap missions of epigenomics and molecular libraries &imaging, developing the HTS assay and identifying PMT inhibitors will vertically advance epigenetic research and facilitate the use of PMT inhibitors for pharmacological intervention. PUBLIC HEALTH RELEVANCE: Deregulated protein methyltransferases (PMTs) have been implicated in many diseases including cancer. The impact of this proposed research is to develop an efficient approach for identifying PMT inhibitors. These molecules can be used as pharmacological probes to investigate the mechanism of PMT-involved diseases. Additionally, novel therapeutic reagents can be developed from PMT inhibitors.